Neutralizing antibodies against HIV infection

ABSTRACT

Neutralizing antibodies against HIV infection are provided. The antibodies are reactive with cryptic epitopes on gp120 and/or CD4 induced by the formation of immunogenic complexes comprising gp120 covalently bonded to CD4 or to succinyl concanvalin A.

This is a divisional of application Ser. No. 08/464,680, filed Dec. 20,1995, now U.S. Pat. No. 5,843,454, which is a national phase ofPCT/US94/05020, filed May 6, 1994, which is a continuation-in-part ofSer. No. 08/060,926, filed May 7, 1993, abandoned.

DESCRIPTION OF THE INVENTION

We discovered that a gp120-CD4 covalently bonded complex presents aspecific subset of cryptic epitopes on gp120 and/or CD4 not present onthe uncomplexed molecules. These complexes elicited neutralizingantibodies with novel specificities and are thus useful in vaccines andimmunotherapy against HIV infection. In addition, the complexes orantibodies thereto can be used in immunological tests for HIV infection.

BACKGROUND OF THE INVENTION

Neutralizing antibodies are considered to be essential for protectionagainst many viral infections including those caused by retroviruses.Since the initial reports of neutralizing antibodies in HIV-infectedindividuals, it has become increasingly clear that high levels of theseantibodies in serum correlate with better clinical outcome (3-5). Thesestudies suggested that the identification of epitopes that elicit hightiter neutralizing antibodies would be essential for vaccine developmentagainst HIV infection.

The primary receptor for the human immunodeficiency virus type 1 (HIV-1)is the CD4 molecule, found predominantly on the surface ofT-lymphocytes. The binding of HIV-1 to CD4 occurs via the major viralenvelope glycoprotein gp120 and initiates the viral infection process.

Current strategies for developing vaccines against infection by thehuman immunodeticiency virus have focused on eliciting antibodiesagainst the viral envelope glycoprotein gp120 or its cell surfacereceptor CD4. Purified gp120 typically elicits type specificneutralizing antibodies that are reactive against epitopes that varyamong virus isolates. This characteristic has hindered the use of gp120as a vaccine.

CD4 has also been considered as a major candidate for development of avaccine against HIV-1. Recent studies have demonstrated that sCD4elicits HIV neutralizing antibodies in animals and prevents the spreadof infection in SIV-infected rhesus monkeys (1). However, autoantibodiesto CD4 may themselves create immune abnormalities in the immunized hostif they interfere with normal T-cell functions. Neutralizing antibodiesagainst gp120 are elicited in vivo in HIV-1-infected individuals and canbe elicited in vitro using purified envelope glycoprotein. However,gp120 contains five hypervariable regions one of which, the V3 domain,is the principal neutralizing epitope. Hypervariability of this epitopeamong strains is a major obstacle for the generation of neutralizingantibodies effective against diverse strains of HIV-1. For these reasonsit has been believed that vaccine strategies using either purified CD4or gp120 present several disadvantages.

We have overcome the shortcomings of type specific anti gp120 antibodiesand antibodies against CD4 by raising anti-HIV-1 neutralizing antibodiesusing as the immunogen a complex of gp120 chemically coupled to eithersoluble CD4 or to the mannose-specific lectin, succinyl concanavalin A(SC). We have found that these compounds induce similar conformationalchanges in gp120. The complexed gp120 appears to undergo aconformational change that presents an array of epitopes that werehidden on the uncomplexed glycoprotein (2). A portion of such epitopeselicit group-specific neutralizing antibodies, which are not strainlimited like the type specific antibodies discussed above. We havediscovered that the covalently bonded CD4-gp120 complexes are useful forraising neutralizing antibodies against various isolates of HIV-1 andagainst HIV-2.

The major research effort in the development of subunit vaccines againstHIV has been directed toward the envelope glycoprotein of the virus.Inoculation of gp160 or gp120 into animals elicits neutralizingantibodies against HIV (3, 4). The principal neutralizing epitope ongp120 has been located between amino acids 306 and 326 in the thirdvariable domain (V3 loop) of the protein (4). This epitope has beenextensively studied by using both polyclonal and monoclonal antibodies(3, 4). In most cases antibodies directed to this region neutralizeHIV-1 in an isolate specific manner although there is evidence that aweakly neutralizing species of anti-V3 loop antibodies can cross-reactwith diverse isolates (8). The reason for type specificity of anti-V3loop antibodies is the extensive sequence variability among variousisolates. Prolonged culturing of HIV-infected cells with type specificanti-V3 loop antibodies induces escape mutants resistant toneutralization (9).

In addition to the V3 loop, other neutralizing epicopes encompassinggenetically conserved regions of the envelope have been identified (10,11). However, immunization against these epitopes elicits polyclonalantisera with low neutralizing titers (12). For example, the CD4 bindingregion of gp120, encompassing a conserved region, elicits neutralizingantibodies against diverse isolates (13). However, this region is notnormally an immunodominant epitope on the glycoprotein.

The interaction of gp120 with CD4 has been studied in considerabledetail and regions of the molecules involved in complex formation havebeen determined (14-16). There are now several lines of evidence thatinteractions with CD4 induce conformational changes in gp120. First,recombinant soluble CD4 (sCD4) binding to gp120 increases thesusceptibility of the V3 loop to monoclonal antibody binding and todigestion by exogenous proteinase (2). Second, sCD4 binding results inthe dissociation of gp120 from the virus (17, 18). These conformationalchanges in gp120 are thought to facilitate the processes of virusattachment and fusion with the host cell membrane (2). Immunization withsoluble CD4 and recombinant gp120, complexed by their natural affinitybut not covalently bonded, resulted in the production of anti CD4antibodies (31). Several murine monoclonal antibodies have beendeveloped by immunization with mixtures of recombinant gp120 and sCD4(31, 32). Antibodies raised in these studies were not strictlycomplex-specific and reacted with free gp120 or CD4; the neutralizingantibodies reacted with free sCD4, although they displayed variousdegrees of enhanced reactivity in the presence of gp120. The complexesused in these studies were unstable and comprised noncovalently boundgp120 and CD4.

A variety of N-linked carbohydrate structures of high mannose, complexand hybrid types present on the gp120 molecule may also play a role inthe interaction of gp120 with host cell membranes (19-21). Indeed, acarbohydrate-mediated reactivity of gp120 has already been demonstratedwith a serum lectin, known as mannose-binding protein, which has alsobeen shown to inhibit HIV-1 infection of CD4+ cells (22). An additionalcarbohydrate-mediated interaction of gp120 has been shown with theendocytosis receptor of human macrophage membranes (21). It has beenpostulated that high affinity binding of accessible mannose residues ongp120 to the macrophage membrane may lead to virus uptake by themacrophage (21).

Recombinant soluble CD4 has been shown to inhibit HIV infection invitro, mainly by competing with cell surface CD4. This observation hasled to the possibility of using sCD4 for the therapy of HIV-infectedindividuals (23, 24). In addition, sCD4 has been used as an immunogen toblock viral infection in animals. Treatment of SIV_(MAC) -infectedrhesus monkeys with sCD4 elicited not only an antibody response to humanCD4 but also to monkey CD4. Coincident with the generation of suchimmunological responses, SIV could not be isolated from the PBL and bonemarrow macrophages of these animals (1). A recent study with chimpanzeesalso demonstrated that human CD4 elicited anti-self CD4 antibody thatinhibited HIV infection in vitro (25). Although immunization with sCD4may be beneficial in blocking HIV infection, circulating antibody thatrecognizes self antigen may induce immune abnormality and dysfunction bybinding to uninfected CD4+ cells. Nevertheless in theory anti-CD4antibodies could be effective in blocking HIV infection provided theycan disrupt virus attachment and entry without interfering with normalCD4 function. Ideally these antibodies should recognize CD4 epitopesthat are present only after interaction with gp120.

SUMMARY OF THE INVENTION

We discovered that gp120-CD4 complex formation induces a specific subsetof cryptic epitopes on gp120 and/or CD4 not present on the uncomplexedmolecules. These epitopes elicit neutralizing antibodies with novelspecificities and are thus useful in vaccines and/or immunotherapy ofpatients infected with HIV. In addition, the antibodies or the complexescan be used in immunological tests for HIV infection. We havedemonstrated that the lectin, SC, mediates changes in the structure ofgp120 in a manner similar to that mediated by CD4. The binding of SC togp120 is another mechanism for inducing novel epitopes on the viralglycoprotein.

We used chemically-coupled gp120-CD4 complexes as immunogens for raisingneutralizing antibodies. We found that gp120-CD4 complexes possess novelepitopes that elicit neutralizing antibodies. Coupling with SC causedperturbation in the gp120 conformation which in turn unmasked crypticneutralizing epitopes on gp120.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the dissociation of gp120 from HIV-1 in the presence ofsCD4 and SC. In FIG. 1A labeled cells were treated with 0 (lanes 1, 2)or 1.5 μg/ml sCD4 (lanes 3, 4). Virus bound (lanes 1, 3) or soluble(lanes 2, 4) gp120 was detected by immunoprecipitation with HIV-1antibody-positive human serum, SDS-PAGE and autoradiography. In FIG. 1Blabeled cells were treated with 0 (lanes 1, 2), 5 μg/ml (lanes 3, 4) or10 μg/ml SC (lanes 5, 6). Virus bound (lanes 1, 3, 5) or soluble (lanes2, 4, 6) gp120 was detected as in 1A.

FIG. 2 illustrates the susceptibility of gp120 to thrombin digestion inthe presence of SC and sCD4. Molt3/HIV-1_(IIIB) cells were labeled with³⁵ S-methionine for 4 hr, followed by a 3 hr incubation with mediumcontaining 0.25% methionine. In FIG. 2A an aliquot of labeled medium (1ml) was digested with thrombin (7 μg/ml) at 37° C. for 90 min and thenimmunoprecipitated with HIV-1 positive human serum and analyzed bySDS-PAGE. Lane 1 shows untreated medium and lane 2, medium treated withthrombin. Prior to thrombin digestion, aliquots of the medium werepretreated with SC at concentrations of 2.5 μg/ml (lane 3), or 10 μg/ml(lane 4); or with sCD4 at concentrations of 2.5 μg/ml (lane 5) or 10μg/ml (lane 6). The gp120 fragments generated by thrombin cleavage aremarked with arrows. In FIG. 2B aliauots of labeled medium were digestedby thrombin as before with no pretreatment (lane 1), after pretreatmentwith 5 μg/ml SC (lane 2 or with a mixture of 5 μg/ml SC and 0.1 mMα-methylpyranoside (lane 3).

FIG. 3 shows the inhibition of HIV-1 induced syncytia formation bymurine antisera raised against gp120-sCD4. In FIG. 3A murine antiserumraised against gp120-sCD4 was added to CEM cells along with cellsinfected with HIV-1_(IIIB) (◯), HIV-1_(MN) (□) or HIV-2_(WAVZ) (⋄). InFIG. 3B murine antisera raised against thrombin treated gp120-sCD4complexes were tested. The assay conditions are described in theExamples. For each experimental condition, the syncytia in threeseparate fields were counted. The average value is given assyncytia/field.

FIG. 4 shows Western blot assays of monoclonal antibodies raised againstgp120-CD4 complexes with gp120, sCD4 and complex. Lane 1 is MoAb7E3,lane 2 is MoAb 8F10E, lane 3 is MoAb 8F10C, lane 4 is MoAb 8F10D, lane 5is anti-gp120 MoAb, lane 6 is anti-p24 MoAb (negative control), lane 7is rabbit anti-CD4 hyperimmune serum, and lane 8 is normal rabbit serum.

FIG. 5 is a graph showing the binding of complex-specific monoclonalantibodies to gp120-lectin complex. MoAbs A(◯) and B(Δ) were tested inELISA, with either gp120-SC (open symbols) or gp120 (closed symbols).

FIG. 6 is a graph showing competitive ELISA with complex-specificmonoclonal antibodies and immune goat serum. Limiting dilutions ofpurified MoAb 7E3 (▪), MoAb 8F10B(◯), MoAb 8F10C() and MoAb 8F10D(▴)were incubated with serial dilutions of goat 69 serum and tested ingo120-CD4 ELISA. Percent competition was calculated as level of antibodybinding in immune serum versus binding in prebleed serum.

FIG. 7 is a photograph of a gel showing gp120-CD4 complexes preparedaccording to Example III. FIG. 4 lane 1 is gp120, lane 2 is sCD4, Lane 3has a gp120-CD4 complex and lane 4 has molecular weight markers.

DETAILED DESCRIPTION OF THE EMBODIMENTS

We determined that it was necessary to unmask or create new epitopes ongp120 and/or CD4 capable of eliciting a strong, broadly neutralizingimmune response. We used a covalently linked gp120-CD4 complex as animmunogen. gp120 molecules were covalently coupled to solublerecombinant CD4 using bivalent cross-linking agents to ensure that theintegrity of the complexes was maintained during any manipulations. Thecomponents of the complex were expected to differ from the freeglycoprotein in at least two ways: (I) some epitopes on gp120 and CD4would be masked by complex formation and (II) cryptic epitopes wouldbecome exposed as a result of conformational changes in gp120 and CD4 ofthe complex. Because these epitopes could play a significant role inviral entry into target cells, antibodies directed against them shouldinhibit some aspects of the entry process. We believed these antibodiesmay not inhibit gp120-CD4 interaction but may instead preventpost-binding fusion events necessary for infection.

The application of this strategy toward anti-HIV vaccines offeredseveral other advantages. First, epitopes specific to complexed gp120are not expected to be normal targets for neutralizing antibodies invivo. HIV-1 binds and enters target cells within 3 min at 37° C. (26).Given the transient and short-lived nature of the native gp120-CD4complex, it is unlikely that it is presented to the immune system insuch a way as to elicit complex-specific antibodies. Therefore, theabsence of immune selection in vivo should in turn be reflected in aminimal degree of variation in the complex-specific epitopes ofdifferent viral strains. Second, antibodies against complex-specificepitopes on CD4 are not expected to elicit anti-self antibodies capableof recognizing uncomplexed CD4 on the surface of normal cells. This isespecially important, since anti-CD4 antibodies can mediate cytotoxiceffects.

In the development of vaccines against HIV, the ability to induce novelepitopes on gp120 in the absence of CD4 would be of considerableadvantage. We discovered this is possible. We have bound amannose-specific lectin, SC, with gp120, which induces a conformationalchange on the glycoprotein that appears to be similar to that observedwith sCD4. The alterations include exposure of the V3 loop to exogenousprotease and dissociation of gp120 from the virus membrane. Therefore,covalently linked gp120-SC complexes are also useful as immunogens forexposing novel epitopes and complex specific antibodies in the absenceof CD4.

The vaccines of the present invention are composed of the complex ofgp120-CD4 or gp120-SC together with an acceptable suspension known inthe vaccine art. Preferably, an adjuvant may be added. The only adjuvantacceptable for use in human vaccines is aluminum phosphate (alumadjuvant), and therefore preferably the vaccine of the present inventionis formulated with an aluminum phosphate gel. See Dolin et al., AnnIntern Med, 1991;114:119-27, which is incorporated herein by reference.The dose of the immunogenic complex for purposes of vaccination isbetween about 40 μg to about 200 μg per inoculation. An initialinoculation may be followed by one or more booster inoculations.Preferably, the vaccination protocol will be the same as protocols nowused in clinical vaccination studies and disclosed in Dolin et al.,supra, and Reuben et al., J Acquired Immune Deficiency Syndrome,1992;5:719-725, also incorporated herein by reference.

It is also contemplated that antibodies raised against the immunogeniccomplexes of the present invention can be used for passive immunizationor immunotherapy. The dosage and number of inoculations of theseantibodies will follow those established in the art for immunization orimmunotherapy with immunoglobulins.

The complexes or antibodies thereto can also be used in a method for thedetection of HIV infection. For instance, the complex, which is bound toa solid substrate or labelled, is contacted with the test fluid andimmune complexes formed between the complex of the present invention andantibodies in the test fluid are detected Preferably, antibodies raisedagainst the is immunogenic complexes of the present invention are usedin a method for the detection of HIV infection. These antibodies may bebound to a solid support or labelled in accordance with known methods inthe art. The detection method would comprise contacting the test fluidwith the antibody and immune complexes formed between the antibody andantigen in the test fluid are detected and from this the presence of HIVinfection is determined. The immunochemical reaction which takes placeusing these detection methods is preferably a sandwich reaction, anagglutination reaction, a competition reaction or an inhibitionreaction.

A test kit for performing the methods mentioned in the precedingparagraph must contain either the immunogenic complex according to thepresent invention or one or more antibodies raised thereto. In the kit,the immunogenic complex or the antibody(ies) are either bound to a solidsubstrate or are labelled with conventional labels. Solid substrates andlabels, as well as specific immunological testing methods are disclosedin Harlow and Lane, "Antibodies, A Laboratory Manual", Cold SpringHarbor Laboratory, 1988, incorporated herein by reference.

EXAMPLES

We conducted several studies to show that new epitopes could be exposedon gp120 and CD4. These studies also demonstrated that neutralizingantibodies could be raised against gp120 after treatment that alteredthe conformation of the glycoprotein.

Example I

a. Conformational Changes in gp120 Induced by Complex Formation with CD4

We analyzed the release of gp120 from the virus surface under variousconditions. Molt3/HIV-1_(IIIB) cells were labeled with 35S-methionine(150 μCi/ml) for 3 hours. The labeled cells were then washed andresuspended in RPMI medium containing cold methionine. The cells werethen cultured for 4 hours in the presence of recombinant sCD4 (DuPont).The cell-free supernatant was collected and then passed through aSephacryl S 1000 column in order to separate virions from free viralproteins. Each of the fractions was treated with detergent,immunoprecipitated with human sera positive for anti-HIV-1 antibodies,and analyzed by SDS-PAGE and autoradiography. The amount of gp120present in the virus and free viral protein fractions was quantitated bya densitometric scan of the autoradiograph. In accordance with previousstudies (17, 18), we observed that treatment of virus with sCD4 clearlyresulted in an increased level of gp120 in the free protein fraction anda coincident decrease in the virus fraction (FIG. 1A), indicating thatthe conformation of gp120 was altered to dissociate it from the virion.

To further investigate how sCD4 alters the conformation of gp120, weconducted studies on thrombin-mediated cleavage of gp120. Digestion ofgp120 by thrombin generates 70 kD and 50 kD products (FIG. 2A). Thiscleavage takes place at the V3 loop. A monoclonal antibody directedagainst an epitope within the loop blocks the cleavage completely. Thethrombin-mediated cleavage at the V3 loop of gp120 is enhanced afterbinding with sCD4. This indicates an increased exposure of the V3 loopon the surface of the protein, which renders it more susceptible toprotease cleavage.

b. Conformational Changes in gp120 Induced by Complex Formation withSuccinyl Concanavalin A

It was previously demonstrated that the incubation of HIV withmannose-specific lectins, such as concanavalin A or succinylconcanavalin A attenuates viral infectivity (27, 28). Incubation of35S-methionine-labeled gp120 with SC resulted in the enhancedsusceptibility of the V3 loop to thrombin digestion (FIG. 2A). Thiseffect was specific, as preincubation of lectin with a-methyl mannosideblocked the enhanced effect completely (FIG. 2B). In addition toincreasing the exposure of the V3 loop, interaction of HIV-1 with SCresulted in a dissociation of gp120 from the viral membrane (FIG. 1B).The degree of such shedding was somewhat less than that observed withsCD4. Nevertheless, these studies clearly indicated that sCD4 and SCalter the conformation of gp120, and in a very similar manner.

C. Immunological Properties of Chemically Coupled gp120-CD4 Complexes

We demonstrated that gp120-sCD4 complexes are immunogenic and capable ofeliciting HIV-1-neutralizing antibodies. An immunoaffinity procedure wasused to purify gp120 from chronically-infected H9/HIV-1_(IIIB) cells.The purified gp120 was then crosslinked to sCD4 (DuPont) using thenoncleavable, water-soluble crosslinker, bis(sulfosuccinimidyl) suberate(BS). Mice were inoculated with the complexes and the immune seraexamined for any effect on HIV-induced syncytium formation. Syncytiumformation induced by HIV-1_(IIIB) and HIV-1_(MN) infected cells wasmarkedly inhibited by the immune sera. A representative inhibition curveof one immune serum is shown in FIG. 3A. Syncytium formation induced bycells infected with the highly related HIV-2 was also inhibited in thepresence of the serum. These results demonstrate that gp120-sCD4complexes are capable of eliciting broadly neutralizing antisera.

We also inoculated mice with complexes comprised of thrombin-digestedgp120 and sCD4. In this case, the gp120 V3 loop was expected to bemodified by protease cleavage. Since V3 has been reported to be theneutralizing epitope on gp120, it has been of interest to determine howsuch cleavage would affect the ability of the complex to elicitneutralizing antibodies. As shown in FIG. 3B, inoculation of mice withthrombin-digested gp120-CD4 complexes elicited antibody capable ofblocking syncytium formation induced by the HIV-1_(IIIB) and HIV-1_(MN)isolates. However, this inhibiting effect was not observed with HIV-2induced syncytium formation.

Our preliminary experiments clearly demonstrated that the covalentlycoupled gp120-CD4 complexes can elicit a broadly neutralizing antibodyresponse. We then undertook to determine whether cryptic epitopes on thecomplex are recognized by the neutralizing antibodies and tocharacterize the epitopes.

Example II Immunological Properties of gp120-CD4 Complex

The glycoprotein gp120 used in the preparation of gp120-CD4 complex waspurified from H9/HIV-1_(IIIB) cells by immunoaffinity chromatography.The cells were lysed in a buffer containing 20 mM Tris (pH 8.2), 0.15 MNaCl, 1.0% Triton X-100, and 0.1 mM PMSF. The lysate was centrifuged at100,000×g for 1 hr. The NaCl concentration in the supernatant wasadjusted to 1 M and the lysate was then reacted with an affinity matrixprepared with human anti-HIV immunoglobulins purified from serum of anHIV-antibody positive subject. The bound antigens were eluted with 50 mMdiethylamine, pH 11.5, and the pH of the eluate was immediately adjustedto 8.0 with Tris HCl. The eluate was extensively dialyzed against 10 mMphosphate buffer (pH 6.5) containing 0.5 M NaCl, 0.1 mM CaCl₂, 1 mMMgCl₂, and 0.2 mM MnCl₂, followed by the addition of Triton X-100 toreach 0.2% by weight solution of the detergent. The dialyzed materialwas then passed through a lentil-lectin column. The glycoproteins wereisolated from the lentil-lectin column by elution with 0.4 Mα-methylmannoside and were then dialyzed against 20 mM Tris HCl (pH 8.2)containing 1 M NaCl and 0.2% Triton X-100. The dialyzed material wasthen applied to an affinity matrix prepared with a mouse monoclonalantibody SVM-25 (U.S. Pat. No. 4,843,011) reactive against gp41 toabsorb gp160 and any gp41 present. The flow-through from the affinitycolumn was dialyzed extensively against 10 mM BES (pH 6.5) containing 1mM EDTA and was loaded on a phosphocellulose column equilibrated withthe same buffer. The column was developed with a linear gradient of0-500 mM NaCl and fractions containing gp120 were pooled, concentrated,and dialyzed against PBS.

The purified glycoprotein was coupled to sCD4 (commercially obtainedfrom duPont) by using bis (sulfosuccinimidyl) suberate (BS) (Pierce) asa crosslinker. For this gp120 and sCD4 were mixed at 1:2 molar ratio inPBS and incubated at 37° C. for 1 hr followed by treatment with 0.5 mMBS at room temperature for 1 hr. The complex was further incubatedovernight at 4° C. The excess BS was blocked with 20 mM Tris-HCl (pH8.0).

Development of gp120-CD4 Complex-Specific Monoclonal Antibodies

Balb/C mice were subjected to six biweekly inoculations of the gp120-CD4complex. The initial inoculum (48 μg per mouse) was emulsified inComplete Freunds Adjuvant and administered by subcutaneous injection. Insubsequent inocula (24 μg/mouse) were emulsified in Incomplete FreundsAdjuvant and were administered by intraperitoneal injection. Two weeksafter the final inoculation the animals were bled and the sera examinedfor HIV-1 neutralizing antibodies by a syncytium blocking assay.Briefly, CEM cells (1×10⁵) were cocultured with HIV-1-infected cells(1×10⁴) in the presence of the test serum and the number of giant cellswere counted after 24-40 hr. Syncytium formation induced byHIV-1_(IIIB) - and HIV-1_(MN) -infected cells was markedly inhibited bythe serum of the mice that was immunized with gp120-CD4 complex.Syncytium formation induced by HIV-2-infected cells was also inhibitedby these sera indicating that gp120-CD4 complexes are capable ofeliciting broadly neutralizing antibodies in mice.

After detection of neutralizing antibodies in mice, the animals receiveda final intraperitoneal form of gp120-CD4 complex in PBS withoutadjuvant. On the fourth day, the animals were sacrificed and the spleenextracted. Splenic lymphocytes were flushed from the spleen with asyringe. The cells (7×10⁷) were fused with 1×10⁷ NS-1 mouse myelomacells (ATCC, Rockville, Md.), overnight in super HT [DMEM containing 20%fetal calf serum (Hyclone), 0.1 M glutamine, 10% NCTC-¹⁰⁹ lymphocyteconditioned medium, 0.5 mM Na-pyruvate, 0.2 U/ml insulin, 1 mMoxalacetic acid, and 100 U/ml penicillin/streptomycin] (GIBCO)containing 40% PEG 1540. The cells are then suspended in super HTcontaining 0.4 μM aminopterin and placed in 96-well plates.

Initially, hybridomas were selected for the production of gp120-CD4complex-specific antibodies. Pooled hybridoma supernatants were testedin the ELISA using gp120, CD4 and gp120-CD4 as antigens. Supernatants ofpools containing complex-specific antibodies were tested individually.Hybridomas of interest were cloned by replating in super HT at a densityof 1 cell/well. Supernatants from cloned hybridomas were further testedby ELISA using gp120-CD4 complexes.

Four hybridomas were selected which secreted immunoglobulindemonstrating a high level reactivity against gp120-CD4 complex andnegligible reactivity with either gp120 or sCD4 in ELISA (Table 1).Notably, one of the monoclonal antibodies, MoAb 7E3, was of the IgAisotype. Immunoglobulins were subsequently purified from the ascitesfluid of each hybridoma and further analyzed by Western blot assay withgp120-CD4 complexes, free gp120, or sCD4. While none of the antibodiesreacted with free gp120 or sCD4, antibodies 7E3 and 8F10B displayed highlevels of reactivity with the complex (FIG. 4) and a low molecularweight fragment of complex. Although antibodies 8F10C and 8F10D reactedstrongly with the complex in ELISA (Table 1), reactivity with thecomplex in Western blot was weak. These results suggest that MoAbs 8F10Cand 8F10D are directed against a set of highly conformation-dependent,complex-specific epitopes that are distinct from the epitopes recognizedby MoAbs 7E3 and 8F10B.

Purified 7E3, 8F10B, 8F10C, and 8F10D immunoglobulins were tested incell-free infection assays using PHA-stimulated peripheral bloodmononuclear cells (PBMCs) and a variety of HIV-1 isolates. As shown inTable 2, none of the antibodies had any significant effect on theinfection of PBMC by the laboratory-adapted strain, HIV-1IIIB. However,antibodies 7E3, 8F10B, and 8F10C neutralized the infection of PBMC by aprimary isolate of HIV-1MN to a significant extent, whereas antibody8F10D had no effect. In contrast to these results, none of theantibodies blocked syncytium formation induced by H9/HIV-1IIIB orH9/HIV-1MN on CEM cells. Our preliminary experiments suggest that theextent of cell-free neutralization by these complex-specific antibodiesmay depend on the infection rate of the isolate. In general, primaryHIV-1 strains with lower infection rates tend to be neutralized moreeffectively than more virulent lab-adapted strains of HIV-1.

To determine whether the complex-specific antibodies bind to the gp120or the CD4 moiety of the complex, we took advantage of our demonstrationthat the mannose-specific lectin, succinyl cona (SC), perturbs theconformation of the glycoprotein in a manner similar to that induced bysCD4 (33). SC and gp120 were cross-linked with BS3 and tested in ELISA.MoAbs 7E3 and 8F10B reacted strongly with the gp120-SC complex (FIG. 5)but did not react with free gp120 or SC. In contrast, antibodies 8F10Cand 8F10D showed only weak binding to the complex. These results suggestthat antibodies 7E3 and 8F10B are directed towards cryptic epitopesexposed on gp120 in response to sCD4 and SC binding.

Immunological Response Against gp120-CD4 Complex in Goats

We have also analyzed the immunogenic response against gp120-CD4 complexin a larger species of animals. An animal (goat 69) was repeatedlyinoculated with 100 μg gp120-CD4 complex in Freund's adjuvant and afterthe fifth inoculation the serum was examined by ELISA for reactivitywith gp120, sCD4 and the complex. Antibodies reactive against both freegp120 and sCD4 were detected in the sera. To determine ifcomplex-specific antibodies were also elicited, the serum was tested incross-competition assays with MoAbs 7E3, 8F10B, 8F10C, and 8F10D.Two-fold serial dilutions of goat 69 serum were incubated with limitingdilutions of each MoAb and tested in gp120-CD4 complex ELISA. As shownin FIG. 6, antibodies in the goat serum were able to block the bindingof all four monoclonal antibodies.

The goat serum was tested for neutralizing antibodies in syncytiumblocking and cell-free infection assays (Table 3). For comparison, serumfrom another animal (goat 58) taken after five inoculations withHIV-1IIIE viral gp120, was also tested. In syncytium assays, goat 69serum reduced syncytium formation ≧80% at titers of 1:640 and 1:80against HIV-1IIIB and HIV-1MN, respectively; goat 58 serum was much lesseffective. Goat 69 serum neutralized cell-free infection of CEM cells byHIV-1IIIB with a titer of 1:80. Again, this titer was significantlyhigher than the titer (1:20) of goat 58 serum. Goat 69 serum alsomediated group-specific neutralization of cell-free infection by primaryisolates HIV-1MN and HIV-1JRFL (Table 3). The neutralizing titer (1:80)was comparable to that of a broadly neutralizing human serum (1:160)tested in parallel; goat 58 serum failed to block HIV-1MN infection evenat <1:20 dilution. Goat 69 serum was retested after removal of anti-CD4antibodies by preabsorption with CEM cells. Removal of such antibodieswas verified by flow cytometric analysis with SupT1 cells which showednearly 90% reduction in cell surface binding. Despite this reduction,the neutralization titer of the absorbed serum was only two-fold less(1:40) than unabsorbed serum, indicating that neutralization is notentirely due to anti-CD4 antibodies.

The results presented in this example indicate that covalentlycross-linked gp120-CD4 complexes possess a number of immunogeniccomplex-specific epitopes. At least a portion of these epitopes resideon the gp120 moiety of the complex. Moreover, some complex-specificepitopes are targets for broadly neutralizing antibodies specificallyeffective against cell-free infection by diverse HIV-1 strains,including primary field isolates targeted toward PBMC. Based on thesefindings, it is possible that the complexes could serve as a protectivevaccine or immunotherapeutic reagent.

Example III Preparation of gp120-CD4 Complex (1:1 Molar Ratio) Free fromAny Uncomplexed CD4

In the immunization protocol described above gp120 and CD4 werecomplexed at a 1:2 molar ratio. As the immunization with this materialresulted in the isolation of anti-CD4 antibodies, we wanted to preparegp120-CD4 complex (1:1 molar ratio) free from any uncomplexed receptormolecules to optimize the conditions for eliciting anti-gp120antibodies. gp120 and CD4 (1:1 molar ratio) were bound at 37° C. for 1hr, reacted with BS for 1 hr at room temperature and then overnight at4° C. After blocking the free crosslinker with Tris buffer (pH 8.0), thesolution was treated with Sepharose coupled to anti-CD4 monoclonalantibody E for 30 min at room temperature. As E binds to an epitope onCD4 involved in the interaction with gp120, this treatment removed anyuncomplexed CD4 present. A gel showing gp120-CD4 complex prepared inthis manner is shown in FIG. 4. It was clear that only the complex withmolecular weight 170 kD and ˜340 kD is evident in the gel. There was nofree gp120 or CD4 present in the preparation.

Example IV

In order to more accurately determine if the immune response togp120-CD4 complexes differs from the responses to the individual complexcomponents, the following experiment was conducted. Separate groups ofmice were inoculated with equal amounts of CD4, gp120 or gp120-CD4complexes. After five inoculations, sera were taken from the animals andanalyzed. As shown in Table 4, all three of the CD4-immunized animalspossessed syncytium blocking seroantibodies effective againstHIV-1_(IIIB) and HIV-1_(MN). All four sera from the complex-immunizedanimals blocked HIV-1_(IIIB) induced syncytia; two of the four alsoblocked syncytia induced by HIV-1_(MN). Overall, neutralizing titers insera from complex-immunized animals was lower than sera fromCD4-immunized animals. Surprisingly, none of the gp120-immunized animalsdisplayed syncytium blocking seroantibodies.

Reactivity with CD4 in ELISA between the CD4-immunized andcomplex-immunized groups was similar (Table 4). The one exception was acomplex-immunized animal (mouse 8) which possessed a titer of anti-CD4antibodies significantly lower than the other animals. Amongcomplex-immunized animals, the level of anti-CD4 reactivity did notcorrelate with syncytium blocking activity; mouse 10 serum was moreeffective in blocking syncytia than mouse 9 serum, even though mouse 9serum had a slightly higher level of anti-CD4 reactivity.

Overall, complex-immunized animals possessed lower titers of anti-V3loop antibodies; such antibodies were virtually absent from mouse 9serum.

Example V

Sera from CD4-immunized and complex-immunized animals were also testedfor reactivity with a variety of synthetic peptides derived from the CD4sequence (Table 5). Although the overall level of anti-CD4 reactivitybetween CD4-immunized and complex-immunized groups was similar (Table4), the patterns of reactivity with linear epitopes differed. While serafrom CD4-immunized animals reacted with peptides derived from theN-terminal portion of CD4 (peptides A and B), such reactivity was absentin sera from complex-immunized animals. This is in accordance with thefact that the N-terminus of CD4 reacts with gp120. The prevalence ofreactivity with a peptide derived from domain 3 of CD4 (peptide D) wasalso reduced among complex-immunized animals relative to CD4-immunizedanimals. Notably, reactivity with a peptide derived from domain 4 of CD4(peptide F) was unique to complex-immunized animals 10 and 11.

The data of Examples IV and V, taken together, indicate that the immuneresponse against gp120-CD4 complexes is unique and different fromresponses to free CD4 and free gp120. Differences in the anti-complexresponse are reflected in 1) a reduced response against the gp120 V3loop; 2) a reduced response against linear epitopes in the CD4N-terminus; 3) an increased response to linear epitopes in CD4 domain 4.It should be noted that the latter epitopes may be hidden in the freeCD4 molecule.

According to the present invention, using gp120-sCD4 complexes asimmunogens, we have been able to raise HIV-1 neutralizing antibodiesthat are complex specific. The results we have obtained with theseantibodies show that covalently coupled gp120-CD4 complexes possessimmunogenic epitopes that are not normally functional in the unboundproteins.

                  TABLE 1                                                         ______________________________________                                        Reactivity of Monoclonal Antibodies Raised                                    Against gp120-CD4 Complexes in ELISA                                                      (OD 450 nm)                                                       Antibody  Isotype CD4        gp120 Complex                                    ______________________________________                                        7E3       IgA.sub.                                                                              .427       .340  >3.0                                       8F10B     IgG.sub.1                                                                             .146       .175  1.5                                        8F10C     IgG.sub.1                                                                             .119       .191  >3.0                                       8F10D     IgG.sub.1                                                                             .208       .202  >3.0                                       anti-gp120                                                                              IgG.sub.1                                                                             .088       >2.0  >2.0                                       anti-CD4  IgG.sub.1                                                                             >3.0       .103  >3.0                                       ______________________________________                                    

The results shown are with hybridoma supernatants, the samespecificities were evident with purified immunoglobulin.

                  TABLE 2                                                         ______________________________________                                        Neutralization of Cell-Free HIV-1IIIB                                         and of HIV-1MN Primary Isolate by gp120-CD4                                   Complex-Specific Monoclonal Antibodies                                        Antibody                                                                      Concentration   % Inhibition.sup.a                                            μg/ml        HIV-1.sub.IIIB                                                                         HIV-1.sub.MN                                         ______________________________________                                        7E3                                                                           100             37       88.7                                                 50              55       69.8                                                 25              0        29.8                                                 8F10B                                                                         100             26.2     67.2                                                 50              6.2      36.3                                                 25              0        28                                                   8F10C                                                                         100             0        75.8                                                 50              0        29                                                   25              0        0                                                    8F10D           17       0                                                    100                                                                           Anti-CD4        100      100                                                  (control)                                                                     50                                                                            ______________________________________                                         .sup.a PHA stimulated PBMC (2 × 10.sup.5 cells) were infected with      either HIV1IIIB or a primary isolate of HIV1MN (50 TCID50) for 18 hr in       the presence of the indicated amounts of purified antibodies. Cells were      then washed and cultured in fresh medium containing the same quantities o     antibodies. The p24 content of the supernatant was determined on day 7 an     the percent inhibition was calculated relative to control assays carried      out in the absence of the antibodies.                                    

                                      TABLE 3                                     __________________________________________________________________________    Neutraling Activity of Sera from Goats Immunized with Either gp120-CD4        Complex or gp120                                                                            Syncytium Blocking.sup.a                                                                Cell-Free Neutralization.sup.b                                      HIV-1 Strain                                                                            HIV-1 Strain/Target Cell                              Serum   Immunogen                                                                           IIIB MN   IIIB/CEM                                                                           MN/PBMC                                                                             JRFL/PBMC                                  __________________________________________________________________________    Goat 69 gp120-CD4                                                                            1:640                                                                               1:80                                                                             1:80 1:80  1:80                                               Complex                                                               Goat 58 gp120 1:20 <1:20                                                                              1:20 <1:20 Not tested                                 Goat 69 gp120-CD4                                                                           <1:25                                                                              <1.25                                                                              Not tested                                                                         1:40  Not tested                                 (Cell Absorbed)                                                                       Complex                                                               __________________________________________________________________________     .sup.a HIV1.sub.IIIBinfected H9 cells were incubated with uninfected CEM      cells in the presence of twofold serial dilutions of each serum. The          number of syncytia were scored in 3 fields of each well after 24 hr.          .sup.b Immune and preimmune serum from each goat was diluted 1:10 in          culture media. The immune serum was then diluted serially in preimmune        serum, thus maintaining a constant serum concentration in all assay wells     Preimmune goat sera and normal human serum did not demonstrate                neutralization relative to control assays in which serum was omitted.         The titers shown produced ≧80% reaction in syncytia or                 neutralization relative to matched preimmune sera.                       

                                      TABLE 4                                     __________________________________________________________________________                Syncytia Blocking                                                             Titer.sup.a HIV-                                                                       HIV-1.sub.B10.sup.V3 Peptide                                                           CD4 ELISA                                       Mouse                                                                              Immunogen                                                                            1.sub.IIIB /HIV-1.sub.MN                                                               ELISA Titer.sup.b                                                                      Titer.sup.c                                     __________________________________________________________________________    1    CD4    1:1600/1:1600                                                                          Not Tested                                                                             >1:256,000                                      2    CD4     1:800/1:1600                                                                          Not Tested                                                                             >1:256,000                                      3    CD4    1:800/1:400                                                                            Not Tested                                                                             >1:256,000                                      4    gp120  <1:50/<1:50                                                                             1:3200  Not Tested                                      5    gp120  <1:50/<1:50                                                                             1:1600  Not Tested                                      6    gp120  <1:50/<1:50                                                                            1:200    Not Tested                                      7    gp120  <1:50/<1:50                                                                             1:3200  Not Tested                                      8    complex                                                                              1:100/<1:50                                                                            1:400       1:32,000                                     9    complex                                                                              1:100/<1:50                                                                            <1:25      1:256,000                                     10   complex                                                                              1:400/1:200                                                                            1:800      1:128,000                                     11   complex                                                                              1:400/1:100                                                                            1:800      1:128,000                                     __________________________________________________________________________     .sup.a Titers are given as the highest serum dilution producing 100%          blocking of syncytia formation. Preimmune sera did not reduce syncytia        relative to control experiments in which serum was absent.                    .sup.b Serial twofold dilutions of each serum was tested. ELISA valves        (absorbance at 450 nm) were converted by subtraction of valves obtained       with the same dilutions of preimmune serum. Titers are given as the           highest serum dilutions having a corrected ELISA valve of ≦0.5.        .sup.c Titers are given as the highest serum dilution having a converted      ELISA valve of ≦0.5.                                              

                                      TABLE 5                                     __________________________________________________________________________    CDR4 Peptide ELISA Valves (A.sub.450nm).sup.a                                 Mouse                                                                             Immunogen                                                                           A  B  C  D  E  F  G  H  I  J  K                                     __________________________________________________________________________    1   CD4   1.58                                                                             1.05                                                                             0.16                                                                             2.65                                                                             0.21                                                                             0.18                                                                             0.35                                                                             0.21                                                                             0.13                                                                             0.15                                                                             0.19                                  2   CD4   2.37                                                                             0.38                                                                             0.17                                                                             2.6                                                                              0.22                                                                             0.17                                                                             0.14                                                                             0.21                                                                             0.15                                                                             0.18                                                                             0.24                                  3   CD4   0.56                                                                             0.29                                                                             0.12                                                                             2.34                                                                             0.18                                                                             0.13                                                                             0.21                                                                             0.19                                                                             0.13                                                                             0.14                                                                             0.20                                  8   Complex                                                                             0.28                                                                             0.27                                                                             0.17                                                                             0.40                                                                             0.23                                                                             0.16                                                                             0.13                                                                             0.19                                                                             0.15                                                                             0.16                                                                             0.26                                  9   Complex                                                                             0.23                                                                             0.29                                                                             0.20                                                                             0.23                                                                             0.21                                                                             0.19                                                                             0.15                                                                             0.20                                                                             0.14                                                                             0.24                                                                             0.17                                  10  Complex                                                                             0.17                                                                             0.33                                                                             0.26                                                                             0.61                                                                             0.3                                                                              1.53                                                                             0.17                                                                             0.36                                                                             0.17                                                                             0.35                                                                             0.13                                  11  Complex                                                                             0.33                                                                             0.43                                                                             0.3                                                                              2.2                                                                              0.36                                                                             0.56                                                                             0.34                                                                             0.36                                                                             0.28                                                                             0.3                                                                              0.20                                  __________________________________________________________________________     .sup.a Sera were tested at a dilution of 1:1000 for reactivity with           peptides derived from the CD4 sequence. Peptide A, residues 25-58; B,         residues 37-53; C, Residues 318-335; D, residues 230-249; E, residues         297-314; F, residues 330-344; G, residues 350-369; H, residues 310-324; I     residues 81-92 (Benzylated); J, residues 81-92; K, irrelevant peptide.        ELISA valves ≧ twofold higher than valves with irrelevant peptide      are shown in Bold type. Reactivity of preimmune  #serum with the CD4          peptides was the same as with the irrelevant peptide.                    

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We claim:
 1. An antibody reactive with cryptic epitopes on gp120 and/orCD4 induced by the formation of an immunogenic complex comprising gp120covalently bonded to CD4.
 2. The antibody of claim 1, which is amonoclonal antibody.
 3. An antibody reactive with cryptic epitopes ongp120 induced by the formation of an immunogenic complex comprisinggp120 covalently bonded to succinyl concanvalin A.
 4. The antibody ofclaim 3, which is a monoclonal antibody.
 5. An immortalized cell linethat produces the antibody of claim
 2. 6. An immortalized cell line thatproduces the antibody of claim
 4. 7. A method for the detection of HIVantigen in a test fluid, comprising contacting the test fluid with anantibody reactive with cryptic epitopes on gp120 and/or CD4 induced bythe formation of an immunogenic complex comprising gp120 covalentlybonded to one of CD4 and succinyl concanavalin A, and detecting thepresence of immune complexes formed between antigen in the test fluidand said antibody.
 8. A test kit for conducting the method of claim 7,comprising said antibody that is bound to a solid substrate or labelledand instructions for performing the detection method.